Current methods of manipulating DNA fragments are each limited by size. Plasmid sized fragments of up to 10 kb can be easily assembled, but specifically ordered fragments much larger are difficult to assemble by current techniques and require careful handling to avoid breakage. Other types of cloning vehicles allow larger fragments to be cloned and manipulated, but even these have their limits (lambda ˜15-20 kb; cosmids ˜35-40 kb, BAC ˜100 kb and YACs ˜1000 kb). Therefore, there is a need in the art for the assembly and manipulation of very large DNA fragments.
PCR fragments can be assembled into larger arrangements for useful purposes. This is usually done with the creation of restriction sites in the primer sequences. When the amplified DNA is cut with a particular restriction enzyme, a short overhang is generated that can be used to assemble two PCR fragments with complementary overhang sequences. However, PCR fragments often have like ends, so that orientation of the resulting fragment is not defined. Further, many restriction enzymes also cleave within a large PCR fragment and cannot be used in this way. When different restriction enzyme sites are used in each of the two amplification primers, the likelihood that one of restriction enzymes will cut within the PCR fragment is doubled. A method that did not depend on restriction enzymes would be of general application and advantageous.
The invention provides a means of assembling PCR fragments that does not rely on restriction enzymes.